It has recently been reported that over 52% of microRNA (miRNA) genes are in cancer-associated genomic regions or in fragile sites. Moreover, an increasing number of studies show that the deregulation of specific miRNAs are linked to different forms of cancer; suggesting that miRNA may have diagnostic potential. Current approaches to measuring miRNA levels, however, lack the sensitivity to detect a small number of cells with altered miRNA expression in the presence of a large number of "normal" cells in human specimens. The overall goal of this proposal is to develop a molecular imaging probe that will allow miRNA expression levels to be measured at the single living cell level via flow cytometry. Since miRNAs are shorter, more stable, and much more abundant than messenger RNAs within individual cells, our plans to target miRNA with 'quantitative molecular beacons' (QMBs) has a significant inherent advantage compared with mRNA-based methods. Moreover, the use of flow cytometry allows for the highly sensitive, high-throughput screening of single cells. Therefore, we hypothesize that the proposed QMB technology could provide an important advance in the early detection of cancer. The specific aims for this proposal are (1) optimize the optical properties and the delivery of QMBs into breast cancer cells and (2) evaluate the sensitivity, precision, and accuracy of the QMB-based approach in detecting aberrant miRNA expression at the single cell level. The long-term goal of this research is to further develop the QMB technology into a simple clinical assay for cancer detection, diagnosis, and prognosis. [unreadable] [unreadable] [unreadable]